The human milk fat globule (HMFG) can be used as a source of antigenic material for the preparation of both polyclonal and monoclonal antibodies for use in the diagnosis and treatment of breast cancer, as well as in the study of the breast epithelial cell surface and the processing of its antigenic components.
The milk fat globule membrane is derived from the apical surface of the mammary epithelial cell during lactation (Patton, S., et al. (1975) Biochim Biophys Acta 415: 273-309). As a result, the HMFG, has been a source for isolation of breast membrane glycoproteins (Taylor, P. J., et al. (1981) Int J Cancer 28: 17-21). Using the HMFG membrane as an immunogen, polyclonal antisera were prepared that proved to have specificity for breast epithelial cells after absorption with non-breast tissue. These polyclonal antisera specifically bound three glycoproteins of molecular weights of 150, 70, and 46 kDa, respectively (Ceriani, R. L., et al. (1977) Proc Natl Acad Sci USA 74: 582-6).
Monoclonal antibodies against the HMFG have been used in the identification of a novel component of the breast epithelial cell surface, a large molecular weight mucin-like glycoprotein, that was named non-penetrating glycoprotein or NPGP (Peterson, J. A., et al. (1990) Hybridoma 9: 221-35; and Ceriani, R. L., et al. (1983) Somatic Cell Genet 9: 415-27). This molecule has been used as a target in breast cancer radioimmunotherapy (Kramer, E. L., et al. (1993) J Nucl Med 34: 1067-74; and Ceriani, R. L., et al. (1988) Cancer Res 48: 4664-72), in the development of a serum assay for breast cancer diagnosis (Ceriani, R. L., et al. (1982) Proc Natl Acad Sci USA 79: 5420-4; and Ceriani, R. L., et al. (1992) Anal Biochem 201: 178-84), and in breast cancer prognosis using immunohistochemistry (Ceriani, R. L., et al. (1992) Int J Cancer 51: 343-54). This non-penetrating glycoprotein (NPGP) appears to be extremely antigenic in mice. The vast majority of monoclonal antibodies prepared against HMFG as well as breast tumors have been found to have specificity against different epitopes of this mucin.
However, the smaller molecular weight proteins of the HMFG also appear to be important surface markers for breast epithelial cells. The 46 kDa and 70 kDa HMFG antigens are also found in the serum of breast cancer patients and thus can be used as markers for breast cancer in serum assays. In addition, the 70 kDa component has been found to co-purify with the intact NPGP complex and has been shown to be linked to NPGP by disulfide bonds.
Few monoclonal antibodies, however, have been prepared against the smaller components of the human milk fat globule system, such as the 70 kDa and 46 kDa HMFG antigens. Although, Peterson, J. A., et al. (1990) Hybridoma 9: 221-35 were able to generate a group of monoclonal antibodies against HMFG that did detect the 46 kDa HMFG antigen, including the Mc3 antibody. The 46 kDa component of the HMFG system, also known as BA46, has been found to be present in the serum of breast cancer patients (Salinas, F. A., et al. (1987) Cancer Res 47: 907-13), and an increase in circulating BA46 was found to be associated with increased tumor burden. In addition, BA46 has been a target molecule in experimental radioimmunotherapy of transplanted human breast tumors in nude mice (Ceriani, R. L. et al. (1988) Cancer Res 48: 4664-72).
In some breast carcinomas, there is an over-expression of the BA46 antigen (Larocca, D., et al. (1991) Cancer Res 51: 4994-8). Also, in human milk BA46 appears to have anti-rotavirus activity that may involve binding to rotavirus (Yolken, R. H., et al. (1992) J Clin Invest 90: 1984-91) and that may interfere with viral infections in newborns.
A partial cDNA sequence of BA46 has been previously reported (Larocca, D., et al. (1991) Cancer Res 51: 4994-8) that placed BA46 in a family of proteins possessing factors V/VIII C1/C2-like domains related to discoidin I (Johnson, J. D., et al. (1993) Proc Natl Acad Sci USA 90: 5677-81). BA46's closest relatives may be found among the murine MGF-E8 (Stubbs, J. D., et al. (1990) Proc Natl Acad Sci U.S.A. 87: 8417-21), the bovine components 15/16 (Mather, I. H., et al. (1993) Biochem Mol Biol Int 29: 545-54) and the guinea-pig GP55 (Mather, I. H., et al. (1993) Biochem Mol Biol Int 29: 545-54) proteins.
cDNA cloning and in vitro cell adhesion studies, provide evidence that BA46 is a breast epithelial cell membrane glycoprotein involved in intercellular interactions. BA46 is localized to the membrane fraction when isolated from breast carcinoma cells (Larocca, D., et al. (1991) Cancer Res 51: 4994-8). BA46 most likely interacts with membrane integrins via its RGD containing EGF-like domain.
Carcinomas result from the carcinogenic transformation of cells of different epithelia. Two of the most damaging characteristics of carcinomas are their uncontrolled growth and their ability to create metastases in distant sites of the host, particularly a human host. It is usually these distant metastases that cause serious consequences to the host, since frequently the primary carcinoma may usually be removed by surgery. The treatment of metastatic carcinomas, that are seldom removable, depends on irradiation therapy and systemic therapies of different natures. The systemic therapies currently include, chemotherapy, radiation, hormone therapy, immunity-boosting pharmaceutical agents and procedures, hyperthermia and systemic monoclonal antibody treatment. In the latter case the antibody proteins can be labeled with radioactive elements, immunotoxins and chemotherapeutic drugs.
Radioactively labeled monoclonal antibodies were initially used with success in lymphomas and leukemia, and recently in some carcinomas. The concept underlying the use of labeled antibodies is that the labeled antibody will specifically seek and bind to the carcinoma and the radioactive element will irradiate the tumor in situ. Since the particles discharged during radioactive decay travel some distance through the tumors it is not necessary that every carcinoma cell bind the labeled antibody. The specificity of the monoclonal antibodies permit the selective treatment of the tumor while avoiding the irradiation of non-malignant tissues. The use of systemic radiation and chemotherapeutic agents without targeting agents produce serious toxic side effects in normal, nonmalignant tissues, making these therapies undesirable for carcinomas and the use of radiolabeled monoclonal antibodies a valid alternative.
Antibodies raised against human epitopes have been used for the diagnosis and therapy of carcinomas. Also known are methods for preparing both polyclonal and monoclonal antibodies. Examples of the latter are BrE-2, BrE-3 and KC-4 (e.g., U.S. Pat. Nos. 5,077,220; 5,075,219 and 4,708,930).